Human cytomegalovirus (HCMV) contains several families of antigenically related glycoprotein complexes, each comprised of two or more individual glycoproteins associated by disulfide bonds. These complexes and their constituent glycoproteins are present in the envelopes of HCMV virions, and are also expressed on the surface of HCMV-infected cells during permissive viral infection.
Convalescent sera from human subjects who have recovered from HCMV infection contain antibodies reactive with these envelope glycoproteins. Moreover, peripheral blood mononuclear cells (PBMCs) from seropositive subjects proliferate when stimulated with HCMV glycoproteins. Therefore, it is likely that these glycoproteins play an important role in protective immune responses, including antibody-mediated neutralization of extracellular virus, B and T cell responses to control or eradicate replicating intracellular virus to prevent cell-to-cell spread, and immune surveillance to prevent reactivation of latent HCMV infection.
Previously described HCMV envelope complexes and their constituent glycoproteins are summarized in Table I, below.
TABLE I __________________________________________________________________________ Human Cytomegalovirus Envelope Glycoproteins and Glycoprotein Complexes Complex(es) MW(daltons) Glycoproteins(s) MW(daltons) Genes(s) __________________________________________________________________________ gCI 130,000- gp130 (I) 130,000 U.sub.L 180,000 gp 93 (I) 93,000 (.344-.360) &gt;450,000 gp55 (I) 55,000 gCII93 93,000 gp 90 (II93) 90,000 U.sub.S gp 52 (II93) 50-52,000 HXLFI-5 gCII200 &gt;200,000 gp200 (II200) &gt;200,000 (.857-.879) gp 90 (II200) 90,000 gp 52 (II200) 50-52,000 gCIII 280,000 gp145 145,000 U.sub.L gp 86 86,000 (.450-.470) Unassociated gp 93.sup.1 93,000 with any complex Egp 48.sup.2 48,000 U.sub.L -ORF3 (0.054-0.064) __________________________________________________________________________ .sup.1 This glycoprotein is distinct from gp93 of the gcI complex. See Kari et al., J. Virol., 60, 345-352, 1986. .sup.2 See the simultaneously filed U.S. Pat. application S.N. 07/328227 entitled "An Early Envelope Glycoprotein or Human Cytomegalovirus (HCMV); inventor Mark F. Stinski.
The family of antigenically related complexes with molecular weights of 130,000-180,000 and &gt;200,000 daltons has been designated as gcI, which contains glycoproteins with molecular weights of 130,000 (gp130), 93,000 (gp93), and 55,000 (gp55). The glycoproteins were originally identified as gA by L. Pereira et.al., Infection and Immunity, 36, 924-932 (1982) (and in U.S. Pat. No. 4,689,225 to Pereira, Aug. 25, 1987) and subsequently designated as gB based on their extensive amino acid sequence homology with the gB glycoproteins of Herpes Simplex virus (HSV) and Epstein-Barr virus (EBV), and gp11 of Varicella zoster virus (VZV).
These gB glycoproteins derive from a single coding region in the unique long (U.sub.L) region of the HCMV genome between map coordinates 0.344 and 0.360 as described by M. Mach et.al., J. Gen. Virol., 67, 1461-1467 (1986). The gB gene may represent a primordial gene conserved among herpes viruses involved in biological functions critical to the evolution and survival of these viruses.
The gB gene was originally detected by expression of a cDNA library in .DELTA.gt11 as detected with a monospecific rabbit antiserum reactive with the 55,000 dalton glycoprotein (gp55) of the gcI complexes. M. Cranage et. al., EMBO J., 5, 3057-3063 (1986) subsequently expressed the gB gene in vaccinia and determined that gp55 was derived from a precursor with a molecular weight of 145,000. Cranage et.al. reported that the gB coding region predicts a polypeptide of 906 amino acids in the AD169 strain of HCMV.
Gretch et.al., J. Gen. Virol., 69, 1205 (1988) subsequently reported that the synthesis of the gcI family of HCMV glycoproteins involves the glycosylation of a 95,000 dalton polypeptide encoded by the gB open reading frame to a stable simple N-linked glycoprotein intermediate of 138,000 daltons, gp138. Alternate proteolytic cleavage at several predicted dibasic protease recognition sites may account for the mature glycoproteins, gp55 and gp93, and possibly for gp130. Recent reports indicate that gp130 detected in detergent extracts of extracellular virions is a unique mature glycoprotein that is distinct from the glycosylated precursor, gp138. Taylor et.al., Antiviral Research, 10, 11-26, 1988.
Spaete et.al., Virology, 167, 207 (1988) reported the amino acid sequence of gB from Towne strain HCMV, which predicts a polypeptide of 907 amino acids with 95% homology to the gB gene in AD169. Analysis of the DNA sequence predicted an N-terminal signal sequence and a C-terminal transmembrane region composed of two domains of hydrophobic amino acids. Eucaryotic expression of the gB gene in COS cells (SV40-transformed simian cells) resulted in the synthesis of a gp130 precursor and a mature gp55 glycoprotein, as determined by reactivity with a neutralizing gcI-specific monoclonal antibody. N-terminal sequencing of purified gp55 showed that this glycoprotein is derived by proteolytic cleavage at dibasic amino acids 460-461 and represents the C-terminal region of gp130. Spaete et.al. further localized one neutralizing epitope to a 186 amino acid fragment of gp55 starting at the N-terminal amino acid 461 by expression of a truncated gB gene.
In sum, the gross characteristics of the gC I, II, and III complexes and corresponding glycoproteins have been partially determined. But, the antigenic regions of the complexes and glycoproteins have not as yet been mapped. Such studies would be useful in the development of vaccines and treatment of HCMV infections. Therefore, it is an object of the present invention to characterize biochemically and immunologically the antigenic regions of gp55 and gp93. In particular, another object is the characterization of the epitopes expressed on those proteins.